Collagen is a common substrate for attachment-dependent mammalian cells, both in the living body and in vitro. Naturally, collagen is secreted by cells as long triple-helical monomer, which polymerizes spontaneously into fibrils and strands, which often have a preferential orientation essential to the function of tissues such as skin, bone and nerve. Cells, in turn, will orient themselves parallel a linear pattern (i.e. fibrils, ridges, or grooves) on the surface to which that are attached. During secretion and deposition as the extracellular matrix, the globular propeptides are cleaved by specific procollagen proteinases, triggering fibril formation, as illustrated in FIG. 1A as reported by M. J. Buehler, Proc Nati Acad Sci US 103, 12285-12290, 2006.
Oriented collagen differs from other cell-adhesion substrate in that linearity is inherent at the molecular level, rather a surface phenomenon. Homogeneous material may be textured or printed with attachment factors, but this topography lies in a two-dimensional plane. Other materials with molecular linearity (e.g. carbon nanotubes) do not have collagen's biocompatibility or capacity to interact with native biomolecules.
The property of linear molecular alignment is important for several reasons. For example, highly oriented collagen cell culture scaffold offers advantage at other ends of the cell differentiation pathway. Cells which remain undifferentiated after division are known as “stem cells” particularly if one of the daughter cells goes on to undergo differentiation (specialization and loss of capacity to generate undifferentiated daughter cells—top level in FIG. 1B. To maintain stem cells in their undifferentiated state while encouraging their proliferation, it requires an environment that protects the cell from physical factors that induce differentiation as well as controlling of chemical factors in the nutrient medium. For example, in the body, stem cells reside in niches such as bone marrow (hematopoietic stem cells) or glandular crypts; the stem cells never leave these habitats.
Collagen matrix in many biological systems has a liquid crystal structure. It is the natural state of the collagen, which provides a long-range orientation. Liquid crystal is a state of matter that is intermediate between the crystalline solid and the amorphous liquid. There are three basic phases of liquid crystals, known as smectic phase, nematic phase, and cholesteric phase as illustrated in FIGS. 2a-2c. FIG. 2a illustrates the smectic phase in which one-dimensional translational order, as well as orientational order exists. FIG. 2b illustrates the nematic phase in which only a long-range orientational order of the molecular axes exists. Cholesteric phase is also a nematic liquid type with molecular aggregates lie parallel to one another in each plane, but each plane is rotated by a constant angle from the next plane, as shown in FIG. 2c, FIG. 3, and FIGS. 4a-4b. The cholesteric phase is a chiral form of the nematic phase. Chiral describes a structural characteristic of a molecule that prevents it from being superposed upon its mirror image. The “twisted plywood model” shown in FIG. 3 is a model of the organization of molecules in a cholesteric structure. This model explains how typical series of arcing patterns observed in sections of cells and tissues result not from authentic curved filaments but originate from the successive molecular orientations found in the twisted plywood arrangement. The model is constructed as follows. The molecular directions are represented by parallel and equidistant straight lines on a series of rectangles, with the orientation of the lines rotating from one rectangle to the next by a small and constant angle. A periodicity is visible wherein each 180° rotation of the molecular directions corresponds to the half-cholesteric pitch P/2. The rotation is chosen to be left-handed, as has been found in all biological twisted materials studied so far. A cholesteric axis is defined by the left-hand rule, the closed first of the left hand indicating the progressive direction of twist and the extended thumb of the left hand pointing in the positive direction of the cholesteric axis. Directly visible on the oblique sides of the pyramid are what appear to be superposed series of parallel nested arcs. The concavities of the arcs are reversed on opposite sides of the model. In biological systems this particular geometry has often been described as twisted plywood. More, background information is provided in Cowin and Doty, Tissue Mechanics, Springer, 2007, herein is incorporated by reference in its entirety, especially page 289-339.
Two major types of twists are found in liquid crystals and their biological analogues and are defined by the disposition of the fibrillar elements either in parallel planes (planar twist) or coaxial cylinders (cylindrical twist) (see FIG. 4). The coaxial cylinders or cylindrical twist are also described as helicoidal. Collagen in the secondary osteons of bone tissue is observed to be in a helicoidal pattern as is cellulose in plant cell walls.
Collagen I can be deposited from solution by a variety of process including casting, lyophilization, electrospinning and other processes well known to one skilled in the art. In most of these procedures, collagen fibers of widely varying diameters and lengths from the micrometer range typical of conventional fibers down to the nanometer range are formed, which provides a mat of interlaced fibers having interstices and pores which provide a suitable foundation for anchoring cells. Owing to their small diameters, electrospun fibers possess very high surface-to-area ratios and are expected to display morphologies and material properties very different from their conventional counterparts occurring in nature. Belamie E. et al. J. Phys. Condens. Matter, 2006, 18, 115-129.
Another technique recently reported uses an inkjet printer capable of printing at high resolution by ejecting extremely small ink drops. This method was described in Nakamura M. et al., Tissue Engineering, 11:1658-1666 (2005) wherein the authors used a biocompatible inkjet head and investigated a feasibility of microseeding with living cells. Living cells are easily damaged by heat; therefore, they used an electrostatically driven inkjet system that was able to eject ink without generating significant heat. Bovine vascular endothelial cells were prepared and suspended in culture medium, and the cell suspension was used as “ink” and ejected onto culture disks. Microscopic observation showed that the endothelial cells were situated in the ejected dots in the medium, and that the number of cells in each dot was dependent on the concentration of the cell suspension and ejection frequency chosen. After the ejected cells were incubated for a few hours, they adhered to the culture disks. While these developments have been made, this technique is limited and has not found widespread use. This technique is somewhat useful for delivering a material (e.g., cells) to a particular area but it cannot maintain and preserve the material's orientation.
All prior art methods of forming collagen films and matrices to date suffer from limitations as do the collagen-based materials formed there from. The main limitation is to maintain and preserve the native liquid crystal structure of collagen-like materials. For example, electrospinning and casting methods cannot preserve a long-range orientation. Collagen based films and matrices cannot mimic the native semi-crystalline structures of the extra-cellular matrix in the living biological systems. Other methods, like, for example, Langmuir-Blodgett method, have limited orientation and poor repeatability.
There are also attempts to produce collagen coating in the controlled matter. For example, U.S. Pat. No. 7,354,627 describes a method for preparing a protein polymer (such as collagen) material involves applying a form of energy such as electrical energy, gravitational energy, thermal energy, or chemical energy to a protein to cause the protein to assemble in a controlled arrangement to form a protein polymer material. U.S. Pat. No. 7,338,517 describes a method of producing a tubular tissue scaffold that comprises a tube wall which includes biopolymer fibrils that are aligned in a helical pattern around the longitudinal axis of the tube where the pitch of the helical pattern changes with the radial position in the tube wall. However, neither patent discloses a collagen layer that mimics the natural pattern and textual of collagen containing tissues.
Accordingly, there is significant need for new collagen-based materials mimicking native structures in living biological systems as well as the reliable and robust methods of producing such materials.